The immune response is a complex defense system that is able to recognize and kill invading organisms such as bacteria, viruses, fungi and possibly also some types of tumor cells. The most characteristic aspects of the immune system are the specific recognition of antigens, the ability to discriminate between self and non-self antigens and a memory-like potential that enables a fast and specific reaction to previously encountered antigens. The vertebrate immune system reacts to foreign antigens with a cascade of molecular and cellular events that ultimately results in the humoral and cell-mediated immune response.
The major pathway of the immune defense commences with the trapping of the antigen by accessory cells such as dendritic cells or macrophages and subsequent concentration in lymphoid organs. There, the accessory cells present the antigen on their cell surface to subclasses of T cells classified as mature T helper cells. Upon specific recognition of the processed antigen the mature T helper cells can be triggered to become activated T helper cells. The activated T helper cells regulate both the humoral immune response by inducing the differentiation of mature B cells to antibody producing plasma cells and control the cell-mediated immune response by activation of mature cytotoxic T cells.
Naturally occurring processes sometimes result in the modulation of immune system cell types. Acquired immunodeficiency syndrome (AIDS) is a devastating infectious disease of the adult immune system which significantly affects cell-mediated immunity. This disease is manifested by profound lymphopenia which appears to be the result of a loss of T-lymphocytes which have the helper/inducer phenotype T4 as defined by the monoclonal antibody OKT4 (Fauci, A., et al. (1984) Annals. Int. Med. 100. 92). Other clinical manifestations include opportunistic infections, predominantly Pneumocystis carinii pneumonia, and Karposi's sarcoma. Other disease states include Severe Combined Immuno Deficiency Syndrome (SCID) wherein T, B or both cell types may be depleted in humans. It is the existence of diseases affecting the immune system, such as AIDS and SCID, which has created the need for animal model systems to study the epitology and potential treatment of such disease states. T lymphocytes recognize antigen in the context of self Major Histocompatibility Complex (MHC) molecules by means of the T cell receptor (TCR) expressed on their cell surface The TCR is a disulfide linked heterodimer noncovalently associated with the CD3 complex (Allison, J. P., et al. (1987) Ann. Rev. Immunol. 5, 503). Most T cells carry TCRs consisting of .alpha. and .beta. glycoproteins. T cells use mechanisms to generate diversity in their receptor molecules similar to those operating in B cells (Kronenberg, M., et al. (1986) Ann. Rev. Immunol. 4, 529; Tonegawa. S (1983) Nature 302, 575). Like the immunoglobulin (Ig) genes, the TCR genes are composed of segments which rearrange during T cell development. TCR and Ig polypeptides consist of amino terminal variable and carboxy terminal constant regions. The variable region is responsible for the specific recognition of antigen, whereas the C region functions in membrane anchoring and in transmitting of the signal that the receptor is occupied, from the outside to the inside of the cell. The variable region of the Ig heavy chain and the TCR .beta. chain is encoded by three gene segments, the variable (V), diversity (D) and joining (J) segments. The Ig light chain and the TCR .alpha. chain contain variable regions encoded by V and J segments only.
The V, D and J segments are present in multiple copies in germline DNA. The diversity in the variable region is generated by random joining of one member of each segment family. Fusion of gene segments is accompanied by insertion of several nucleotides. This N-region insertion largely contributes to the diversity, particularly of the TCR variable regions (Davis and Bjorkman (1986) Nature 334, 395), but also implies that variable gene segments are often not functionally rearranged. The rearrangement of gene segments generallyoccurs at both alleles. However, T and B cells express only one TCR or Ig respectively and two functionally rearranged genes within one cell have never been found. This phenomenon is known as allelic exclusion.
During B cell development the rearrangement process starts at both heavy chain gene alleles. First a D segment is fused to a J segment followed by ligation of a V segment to the DJ join. If the VDJ joining is productive, further rearrangement of the other heavy chain allele is blocked, whereas rearrangement of the light chain loci is induced (Reth, M., et al. (1985) Nature 317, 353).
In both B and T cells, partially (DJ) and completely (VDJ) rearranged genes reportedly are transcribed giving rise to two differently sized RNA molecules (Yancopoulos, G., et al. (1986) Ann. Rev. Immunol. 4, 339; Born, W., et al. (1987) TIG 3, 132). In B cells the DJ transcripts can be translated into a D.mu.-chain, a truncated form of the Ig.mu. heavy chain that lacks a V segment derived sequence. In general, the D.mu.-chain is present in minor amounts, if at all. However, in one subclone (P4-11) of the 300-19 cell line, a transformed pre-B cell line which differentiates in vitro to Ig producing B cells, the expression of the D.mu.-chain is reportedly very high (Reth, M., et al. (1985) Nature 317, 353). This reference also reports that the heavy chain gene alleles in the P4-11 clone are blocked at the DJ rearrangement stage in cell culture and that such cells show a very high frequency of light chain gene rearrangements. This has led to the suggestion that the D.mu. protein contains some of the regulatory determinales necessary for gene assembly (Yancopoulos, G., et al. (1986) Ann. Rev. Immunol. 4, 339, 356).
Transgenic mice containing functionally rearranged Ig genes reportedly have been used in studying several aspects of Ig gene expression, e.g. tissue specific expression, the mechanism of segment rearrangement, allelic exclusion and repertoire development (Storb, U. (1987) Ann. Rev. Immunol. 5, 151). It has also been reported that the transgenic heavy chain polypeptide only inhibits the complete VDJ rearrangement of endogenous heavy chain genes if it contains a transmembrane domain (Storb, 1987; Iglesias, A., et al. (1987) Nature 330, 482; Nussenzweig, M., et al. (1987) Science 236, 816; Nussenzweig, M., et al. (1988) J. Exp. Med. 167, 1969).
Recently, the inventors reported that functionally rearranged TCR.beta. genes can be appropriately expressed in transgenic mice (Krimpenfort, P., et al. (1988) EMBO 7, 745). This functional TCR.beta. chain gene prevents expression of endogenous genes .beta. genes by inhibiting complete VDJ joining (Uematsu, Y., et al. (1988) Cell 52, 831).
Two different types of T cells are involved in antigen recognition within the self MHC context. Mature T helper cells (CD4.sup.+ CD8.sup.-) recognize antigen in the context of class II MHC molecules, whereas cytotoxic T cells (CD4.sup.- CD8.sup.+) recognize antigen in the context of class IMHC determinants (Swain, S. L. (1983) Immun. Rev. 74, 129-142; Dialynas, P. D., et al. (1983) Immun. Rev. 74, 29-56). It has been reported that class II- specific CD4.sup.+ CD8.sup.- helper T cells (also referred to as T4 cells) fail to develop in mice neonatally treated with anti-class II MHC monoclonal antibody (Kruisbeek, A. M., et al. (1983) J. Exp. Med. 157, 1932-1946; Kruisbeek, A. M., et al. (1985) J. Exp. Med. 161, 1029-1047). Similarly, it has recently been reported that mice chronically treated with anti-class I MHC monoclonal antibody from birth have a significantly reduced population of CD4.sup.- CD8.sup.+ cells and cytotoxic T cell precursors (Marusic-Galesic, S., et al. (1988) Nature 333, 180-183). Although selected T cell populations apparently can be produced by such methods, continuous administration of antibody is required which often results in adverse side effects in such mice.
A different strategy to deplete specific cell lines has recently been identified wherein specific cell destruction is induced by administration of a toxic metabolite. Specifically, transgenic mice reportedly were produced containing a Herpes Simplex Virus Thymidine Kinase (HSV-TK) transgene fused to the Ig promoter/enhancer. Transgenic cells that express the HSV-TK are not affected. However, upon administration of a nucleoside analog that can be phosphorylated by the transgenic HSV-TK gene, replicating cells expressing the HSV-TK gene are killed (Heyman, et al. (1988) UCLA Symposia on Molecular and Cellular Biology, 73. 199.
Another approach to depletion of specific cell types has been reported using tissue specific expression of a bacterial toxin. Specifically, mice carrying an elastase/diptheria toxin A (DT-A) fusion gene lacked a normal pancreas (Palmeter, et al. (1987) Cell 50, 435). In addition, it has been reported that microphtalmia in transgenic mice resulted from the introduction of the DT-A gene fused to the .alpha.2-crystallin promoter (Bretman, et al. (1987) Science 238, 1563).
Transgenic mice reportedly have also been constructed that express an .alpha..beta.TCR in a large fraction in their T cells which is specific for a minor histo-compatibility antigen (H-Y) present on male, but not female, cells (Kisielow, P., et al. (1988) Nature 333, 742-746). This very recent reference reports that cells containing the TCR for the H-Y antigen were frequent in female but not in male transgenic offspring. The .alpha..beta. TCR in such trasgenic mice apparently contains all the segments and regions required for a functional TCR.
The references discussed above are provided solely for the disclosure prior to the filing date of the present application and nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosures by virtue of prior invention.
Given the state of the art, it is apparent that a need exists for animal model systems to study diseases which effect the immune system including infectious diseases such as AIDS. Accordingly, it is an object herein to provide transgenic non-human animals and methods for making the same which have a phenotype characterized by the substantial depletion of a mature lymphocytic cell type otherwise naturally occurring in the species from which the transgenic is derived.
It is also an object herein to provide transgenic non-human animals substantially depleted in mature T cells or plasma cells.
It is a further object herein to provide transgenic mice substantially depleted in mature T cells or plasma cells.
Still further, it is an object herein to provide transgenes capable of producing such transgenic non-human animals.
Further, it is an object herein to provide methods for producing transgenic non-human animal having at least one of the above identified phenotypes.